Spheroid cell culture article and methods thereof

ABSTRACT

A spheroid cell culture article including:
         a frame having a chamber including:
           an opaque side wall surface;   a top aperture;   a gas-permeable, transparent bottom; and   optionally a chamber annex surface and second bottom, and at least a portion of the transparent bottom includes at least one concave arcuate surface, is disclosed. Methods of making and using the article are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. §111 as a continuationapplication of U.S. patent application Ser. No. 14/087,906, filed onNov. 22, 2013, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/817,539 filed on Apr. 30, 2013, the content of whichis relied upon and incorporated herein by reference in its entity.

The entire disclosure of any publication or patent document mentionedherein is incorporated by reference.

BACKGROUND

The disclosure generally relates to a cell culture well article andmethods of making and using the article

SUMMARY

In embodiments, the disclosure provides a well article for culturing andassaying, for example, spheroidal cell masses. The well article includesat least one chamber having an opaque side wall; a transparent, round orconcave bottom surface; and the transparent bottom is gas-permeable.

In embodiments, the disclosure provides methods for making the wellarticle, and methods of using the well article in spheroid cell cultureor in cellular assays.

In embodiments, the disclosure also provides a multi-well plate articlehaving wells or chambers with opaque walls and a gas-permeable,transparent round-bottom, and methods for making the round-bottommulti-well plate article. The transparent round-bottom, such as anoptically clear bottom window, permits convenient microscopicvisualization or examination of the cultured spheroid cell mass.

In embodiments, the disclosure provides a well plate having a base, thebase having a conical or tapered geometry, such as a forty five degree(45°) angle, or like taper angles, from the sidewall to a radius at theapex (i.e., the very bottom of the well) to provide a round-bottom wellor multiwell plate having an opaque sidewall and an optically clearwindow or base for optical visualization, such as with a microscopic orlike devices, of a spheroid cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

In embodiments of the disclosure:

FIG. 1 schematically shows methods for joining preformed or moldedcomponents to make examples of the disclosed articles.

FIGS. 2A to 2C show an alternative method for making a disclosed articlehaving an opaque sleeve or chamber wall surface and a pre-formed clearbase insert.

FIG. 3 shows another alternative method of making the disclosed articleusing distortion printing.

FIG. 4 shows in a cross-section view of a single well or single chamberin the frame of an alternative exemplary article (400).

FIG. 5 shows in a partial top or plan view aspects of the exemplaryarticle of FIG. 4.

FIG. 6 shows in perspective aspects of an exemplary perfusion plateapparatus (600).

FIG. 7 shows in a cross-section view aspects of an exemplarymulti-spheroidal well article (700).

FIG. 8 shows in a partial top or plan view aspects of the exemplaryarticle of the multi-spheroidal well article (700) of FIG. 7.

FIGS. 9A and 9B show a cross-section view of the combination (900) ofthe injection molding pin shut-off in contact with a well-bottom havingdifferent geometries.

FIGS. 10A and 10B show cross-section views of an alternative design fora pin shut-off.

FIGS. 11A to 11D show images of cell spheroids in alternativewell-bottoms having different geometries.

DETAILED DESCRIPTION

Various embodiments of the disclosure will be described in detail withreference to drawings, if any. Reference to various embodiments does notlimit the scope of the invention, which is limited only by the scope ofthe claims attached hereto. Additionally, any examples set forth in thisspecification are not limiting and merely set forth some of the manypossible embodiments of the claimed invention.

In embodiments, the disclosed apparatus and the disclosed method ofmaking and uuing the apparatus provide one or more advantageous featuresor aspects, including for example as discussed below. Features oraspects recited in any of the claims are generally applicable to allfacets of the invention. Any recited single or multiple feature oraspect in any one claim can be combined or permuted with any otherrecited feature or aspect in any other claim or claims.

DEFINITIONS

“Include,” “includes,” or like terms means encompassing but not limitedto, that is, inclusive and not exclusive.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, viscosities, and like values, and rangesthereof, or a dimension of a component, and like values, and rangesthereof, employed in describing the embodiments of the disclosure,refers to variation in the numerical quantity that can occur, forexample: through typical measuring and handling procedures used forpreparing materials, compositions, composites, concentrates, componentparts, articles of manufacture, or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of a composition or formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a composition or formulation with a particularinitial concentration or mixture.

“Optional” or “optionally” means that the subsequently described event,circumstance, or structure, can or cannot occur, and that thedescription includes instances where the event, circumstance, orstructure, occurs and instances where it does not.

“Consisting essentially of” or “consisting of” in embodiments can referto, for example:

a spheroid cell culture article having:

-   -   a frame having a chamber or a plurality of chambers, for        example, a well, each chamber having:    -   an opaque side wall surface, such as a well having an opaque        side wall;    -   a top aperture for operational access to the chamber;    -   a gas-permeable, transparent bottom surface; and    -   optionally a porous membrane insert in at least one of the        chambers,        at least a portion of the transparent bottom includes at least        one concave arcuate surface, or a plurality of concave arcuate        surfaces within a single chamber;

a method of making the abovementioned spheroid cell culture articleincluding:

-   -   combining the gas-permeable, transparent arcuate bottom surface        portion and an opaque side wall surface portion to form the        article, wherein the combining is accomplished with any of the        methods disclosed herein or any other suitable methods; and    -   optionally inserting a porous membrane liner in at least one of        the chambers; and

a method of using the article for culturing spheroids including:

-   -   charging the disclosed spheroid cell culture article with        culture media; and    -   adding spheroid forming cells to the culture media, as defined        herein.

The article, the method of making the article, and the method of usingthe article, of the disclosure can include the components or stepslisted in the claim, plus other components or steps that do notmaterially affect the basic and novel properties of the compositions,articles, apparatus, or methods of making and use of the disclosure,such as a particular article configuration, particular additives oringredients, a particular agent, a particular structural material orcomponent, a particular irradiation or temperature condition, or likestructure, material, or process variable selected.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Abbreviations, which are well known to one of ordinary skill in the art,may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” forgram(s), “mL” for milliliters, and “rt” for room temperature, “nm” fornanometers, and like abbreviations).

Specific and preferred values disclosed for components, ingredients,additives, dimensions, conditions, and like aspects, and ranges thereof,are for illustration only; they do not exclude other defined values orother values within defined ranges. The apparatus and methods of thedisclosure can include any value or any combination of the values,specific values, more specific values, and preferred values describedherein, including explicit or implicit intermediate values and ranges.

The formation of uniformly-sized spheroids of, for example, tumor cells,can be facilitated if the cells are cultured in a round-bottom vesselthat has low-adhesion properties. Researchers from the Institute ofCancer Research in Sutton, UK, used Corning, Inc.'s, ultra-low-adhesion(ULA)-coated 96-well clear, round-bottom, polystyrene microplates toproduce tumor spheroids that enabled target validation and drugevaluation (see Vinci et al., BMC Biology, 2012, 10:29). The spheroidsenhanced the biological relevance of the tumor cell cultures andfacilitated a range of functional assays. The round-bottom well shapealong with gravity and the ultra-low attachment coat or coating,encouraged the cells to come together and self-assemble into thespheroid shape rather than to form a monolayer of cells.

While the clear, round-bottom, ULA-coated wells of the 96-well platewere utilized with good results for migration and invasion assays due tothe optical clarity of the structural polymer, evaluation of anti-canceragents using fluorescent or luminescent detection in this plate wereless optimal since the clear sidewalls added to the noise in thedetected signal. While the tumor cells could be moved into opaque96-well plates, this calls for pipetting the spheroids, which can bedelicate, difficult, and time-consuming.

In embodiments, the present disclosure provides a spheroid cell culturearticle comprising:

-   -   a frame having a chamber, for example, a well, comprising:        -   an opaque side wall surface;        -   a top aperture; and        -   a gas-permeable, transparent bottom surface,            and at least a portion of the bottom surface comprises at            least one concave arcuate surface, that is, a rounded or            curved surface.

The disclosed well plate having a well base having a conical or taperedgeometry is superior for injection molding of the well's opaque sidewall since it is unnecessary to alter existing flat pins that have beenpreviously used to over-mold on a flat insert (i.e., base). Injectionmolding with existing flat pins can also be accomplished on awell-bottom geometry having a full radius (i.e., a hemispherical oruntapered base). An optional distortion collar feature on the base canenhance the ability of an existing flat pin to achieve shut-off on thewell-bottom geometry.

A well plate having the disclosed tapered or chevron shaped (e.g., a 45degree angle from the sidewall to a radiused apex) well-bottom geometryhas been successfully demonstrated for use in the formation of cellspheroids. The tapered well-base or well-bottom geometry also enablesover-molding of the opaque well wall onto an insert (base) pre-formedwith the tapered geometry using existing flat pins that would ordinarilybe used to over-mold on a flat insert. The optional distortion collarfeature integral with or attached to the insert (base) can facilitateresin flow shut-off of the injection molding pin. The well-bottomgeometry can alternatively be generated by thermal-forming a polymerfilm prior to over-molding, or after over-molding using a thermalreforming process. Thermal reforming permits rapid change-over togenerate well geometries for alternative multiple formats.

The disclosed well plate having a tapered or chevron shaped base (e.g.,an approximate 45 degree angle) is advantaged by, for example,permitting over-molding onto the insert using the same pin equipment toover-mold on a flat insert. This over-molding can be facilitated by theaddition of a distortion collar that can flex to permit the pin toshut-off the polymer flow. Using the flat pin for molding results insaving time and reducing cost for the generation of this well plateproduct since the mold-cavity pins do not need to be modified fordifferent well geometries or different well count formats.

Thermal reforming permits rapid change-over to generate well geometriesfor many different well formats. Thermal reforming also permits designflexibility for assessment of different well geometries for each formatand the ability to rapidly produce prototype or production samples forcustomer evaluation or specification.

A well base having an approximate 45 degree tapered angle also allowsfor a smaller radiused apex, which apex centers the collection of cellsthat settle by gravity to form a cell spheroid, and enhances opticalimaging.

Referring to the Figures, FIG. 4 shows in a cross-section view anexemplary article (400) having a chamber (410), and a chamber annex(420) for receiving an aspirating pipette (440), and optionally a porousmembrane (480), for example, a high throughput screening membrane insertor liner, for dividing the chamber into upper (410 a) and lower (410 b)chambers, or upper and lower chamber volumes. The chamber annex (420) orchamber extension is not physically separated from the main chamber(410) space, but is instead a spatial extension or expansion of the mainchamber. Thus, dotted line (425) represents a non-physical boundarybetween the main chamber and the chamber annex. The chamber annex andthe optional porous membrane provide an excellent geometry that permitsaspiration of the medium without aspirating the spheroid (450) mass fromthe well chamber. The chamber annex (420) having an optional secondbottom (430) or stop-ledge provides space to accommodate a pipette tip(440) of a pipette to exchange fluid medium without significantlydisturbing the spheroid (450) in the transparent arcuate round bottomsurface area and volume (460) of the lower chamber (410 b). The stopledge (430) prevents the pipette tip from entering the lower spheroidchamber.

FIG. 5 shows aspects of the exemplary article of FIG. 4. FIG. 5 shows inthe well area A1, a partial top or plan view (500) of the exemplaryarticle of FIG. 4, having a plurality of the chambers (410), eachchamber having a chamber annex (420) for receiving the aspiratingpipette (440). Additionally or alternatively, FIG. 5 shows in well areaB1, the chamber (410) includes the optional porous membrane (480) orliner for dividing the chamber into the upper chamber and the lowerchamber (410 a and 410 b, respectively, not shown) where the porousmembrane (480) covers the underlying or hidden spheroid (450). Anarticle having wells configured as shown in area B1 including the porousmembrane (480) provide a functioning nested 96-well HTS Transwell®spheroid plate.

Well plates having opaque sidewalls and gas permeable, rounded-bottomwells having clear windows can provide significant advantages including,for example: no need to transfer the spheroid from one multiwell plate(in which spheroids can be formed and visualized) to another plate forconducting assays to, for example, evaluate drug compounds; avoiding thespheroid transfer step can save time and avoid potential loss ordisruption of the spheroid; and the spheroids can receive superioroxygenation with well-bottoms made from a polymer having gas permeableproperties at a given wall thickness. Increased oxygen availability tothe cells in the spheroid culture is particularly helpful for cells withhigh oxygen requirements such as hepatocytes.

In embodiments, the article having a chamber having at least one concavearcuate surface can include, for example, a plurality of adjacentconcave arcuate surfaces having, for example, from 1 to about 1,000concave arcuate surfaces on the bottom of the same chamber, see forexample, FIGS. 7 and 8.

In embodiments, the article can be, for example, a single well ormulti-well plate configuration having numerous “spheroidal wells”, suchas a plurality of dimples or pits in the bottom or base of each well.The plurality of spheroids or spheroid wells per chamber can preferablyaccommodate, for example, a single or one spheroid per spheroid well.

In embodiments, the gas permeable, transparent well bottom having the atleast one concave arcuate surface or “cup” can be, for example, ahemi-spherical surface, a conical surface having a rounded bottom, andlike surface geometries, or a combination thereof. The well and wellbottom ultimately terminates, ends, or bottoms-out in a spheroid“friendly” rounded or curved surface, such as a dimple, a pit, and likeconcave frusto-conicial relief surfaces, or combinations thereof.

In embodiments, the opaque side wall surface (i.e., a surround) can be,for example, a vertical cylinder or shaft, a portion of a vertical conicof decreasing diameter from the chamber top to the chamber bottom, avertical square shaft or vertical oval shaft having a conicaltransition, i.e., a square or oval at the top of the well, transitioningto a conic, and ending with a bottom having at least one concave arcuatesurface, i.e., rounded or curved, or a combination thereof. Otherillustrative geometric examples include holey cylinders, holey coniccylinders, first cylinders then conics, and other like geometries, orcombinations thereof.

In embodiments, the article can further comprise, for example, alow-adhesion or no-adhesion coating on a portion of the chamber, such ason the at least one concave arcuate surface.

In embodiments, the article can further comprise, for example, a chamberannex, chamber extension area, or an auxiliary side chamber, forreceiving a pipette tip for aspiration, the chamber annex or chamberextension (e.g., a side pocket) can be, for example, an integral surfaceadjacent to and in fluid communication with the chamber. The chamberannex can have a second bottom spaced away from the gas-permeable,transparent bottom. The chamber annex and the second bottom of thechamber annex can be, for example spaced away from the gas-permeable,transparent bottom such as at a higher elevation or relative altitude.The second bottom of the chamber annex deflects fluid dispensed from apipette away from the transparent bottom to avoid disrupting ordisturbing the spheroid.

In embodiments, the article can further comprise a porous membrane, suchas a liner or membrane insert, situated within a portion of the chamber,situated within a portion of the chamber annex, or both the chamber andthe chamber annex portion. The porous membrane can provide isolation orseparation of a second cellular material, such as a different cell typeor different cell state, situated in an upper portion of the chamber, inan upper portion of the chamber formed by the porous membrane, or bothchambers, from first cellular material in a lower portion of one or bothchambers near the transparent bottom.

In embodiments, the at least one concave arcuate surface can be, forexample, a hemisphere, or a portion of a hemisphere, such as ahorizontal section or slice of a hemisphere, having a diameter of, forexample, from about 250 to about 5,000 microns (i.e., 0.010 to 0.200inch), including intermediate values and ranges, depending on, forexample, the well geometry selected, the number of concave arcuatesurfaces within each well, the number of wells in a plate, and likeconsiderations. Other concave arcuate surface can have, for example,parabolic, hyperbolic, chevron, and like cross-section geometries, orcombinations thereof.

In embodiments, the spheroid can be, for example, substantially asphere, having a diameter of, for example, from about 100 to about 500microns, more preferably from about 150 to about 400 microns, even morepreferably from about 150 to about 300 microns, and most preferably fromabout 200 to about 250 microns, including intermediate values andranges, depending on, for example, the types of cells in the spheroid.Spheroid diameters can be, for example, from about 200 to about 400microns, and the upper diameters being constrained by diffusionconsiderations (for a review of spheroids and spheroid vessels seeAchilli, T-M, et. al. Expert Opin. Biol. Ther. (2012) 12(10)).

In embodiments, the disclosure provides a perfusion plate apparatuscomprising:

at least one cell culture article comprising:

-   -   a frame having a chamber, for example, a well, comprising:    -   an opaque side wall surface;    -   a top aperture;    -   a gas-permeable, transparent bottom surface; and    -   optionally a chamber annex surface,

the at least a portion of the bottom comprises at least one concavearcuate surface, that is, a rounded or curved surface,

media source well in fluid communication with at least one chamber ofthe cell culture article that controllably provides a source of freshmedia to at least one chamber; and

a waste well in fluid communication with the at least one chamber of thecell culture article that controllably receives waste media from cellmetabolism in the at least one chamber.

FIG. 6 shows in perspective aspects of an exemplary perfusion plateapparatus (600). In embodiments, the aforementioned perfusion plateapparatus can further comprise a porous liner (480) situated within aportion of the chamber (610), situated within a portion of the chamberannex (not shown), or both the chamber and the chamber annex, the porousliner provides isolation or separation of a second cellular material(660) such as Caco 2 cells, in an upper portion of the chamber, an upperportion of the chamber annex, or both upper portion of the chamber andthe chamber annex, from first cellular material, such as a spheroid(450), situated in the lower portion of the chamber or the chamber annexand near the transparent bottom region(460).

In embodiments, the aforementioned perfusion plate apparatus can furthercomprise, for example: a source liquid (not shown), a perfusion plug(655), i.e., liquid permeable material, situated in a liquid connectionmember (635), such as a tube, between a source well (630) and the cellculture rounded-bottom well of the apparatus (600), a perfusion plug(655) situated in a liquid connection member (645) between the cellculture rounded-bottom well and the waste well (640), or bothsituations. The waste well (640) preferably can have an arcuate bottomsurface to facilitate removal of waste liquid medium (not shown) using,for example, a pipette, a vacuum pump, or like liquid removal equipment.

A related but distinct cell culture article (i.e., FloWell™ plate) isdisclosed in commonly owned and assigned copending application, U.S.Provisional Patent Application Ser. No. 61/594,039, filed on Feb. 2,2012, now PCT/US13/24030, to Goral, et al., entitled “Automaticcontinuous perfusion cell culture microplate consumables”. The relatedapplication mentions, for example, a microplate for culturing cells withautomatic, continuous perfusion of a liquid medium, and can include, forexample, a well frame which defines a plurality of cavitiestherethrough. The microplate can further include a planar substrateconnected with the well frame. The planar substrate can provide a bottomsurface to the plurality of cavities, forming a plurality of wells. Theplurality of wells may include a first well, a second well fluidlyconnected with the first well, and a third well fluidly connected withthe first well. The first well may be employed for culturing the cellsin the liquid medium. The second well may be employed for providing anoutflow or source of the liquid medium to the first well. The third wellmay be employed for receiving an inflow or waste stream of the liquidmedium from the first well. The second well may be fluidly connectedwith the first well with a first perfusion membrane. A first perfusionmembrane can be disposed in between the well frame and the planarsubstrate and may extend from an outlet section of the second well to aninlet section of the first well. The third well may be fluidly connectedwith the first well with a second perfusion membrane. The first andsecond perfusion membranes can have a porosity of, for example, fromabout 0.2 to about 200 microns. The second perfusion membrane can bedisposed in between the well frame and the planar substrate and extendfrom an outlet section of the first well to an inlet section of thethird well. Upon introduction of a perfusion-initiating amount of theliquid medium into the second well, the liquid medium flows from thesecond well through the first perfusion membrane to the first well andfrom the first well through the second perfusion membrane to the thirdwell (see for example FIG. 5 therein). The related application alsomentions methods of fabricating the microplate and methods of culturingcells.

FIG. 7 shows aspects of an exemplary multi-spheroidal well article(700), which is reminiscent of aspects of FIG. 4 including, for example,one or more chambers (410), each chamber having the chamber annex (420)area bounded by line (425) and stop-ledge (430), for receiving anaspirating pipette (not shown). Unlike FIG. 4, the bottom of eachchamber (410) in FIG. 7 can include a plurality of arcuate dimples(465), which dimples can be either or both transparent and gaspermeable, and can each receive or cultivate a single spheroid (450).Cells can be seeded into the wells of the apparatus at a low density topermit single spheroid formation and cultivation in the neighboring butseparated cups. The multi-spheroidal well article (700) can include theoptional porous membrane (480) or liner insert for dividing the chamberinto the upper (410 a) and the lower (410 b) chamber.

FIG. 8 shows aspects of the exemplary article of the multi-spheroidalwell article (700) of FIG. 7. FIG. 8 shows, in the well area A1, a topview (800) of the exemplary article of FIG. 7, having a plurality of thechambers (410), each chamber having the chamber annex (420) forreceiving the aspirating pipette (not shown). The bottom of each chamber(410) includes a plurality of arcuate dimples (465), which dimpledsurfaces can receive or cultivate a plurality of spheroids (450).Additionally or alternatively, FIG. 8 shows, in the well area B1, thechamber (410) includes the optional porous membrane (480) or linerinsert for dividing the chamber into the upper chamber and the lowerchamber (410 a and 410 b, respectively, not shown). The membrane (480)or liner insert conceals and protects the underlying spheroids (450) andspheroid wells (465) from disturbances or disruption from forces oractivity above the membrane, such as during the addition or removal ofliquid medium.

In embodiments, the article having a chamber can include, for example,from 1 to about 2,000 chambers, from 10 to about 1,500 chambers,including intermediate values and ranges, and each chamber is physicallyseparated from any other chamber, and each chamber has a single concavearcuate surface. In embodiments, such a multi-well plate productconfiguration can have, for example, one spheroid per chamber.

In embodiments, the at least one concave arcuate surface can have, forexample, a plurality of adjacent concave arcuate bottom surfaces withinthe same well. In embodiments, a single well or multi-well plateconfiguration can have numerous spheroidal wells, such as dimples orpits in the bottom or base of each well. In embodiments, the article canaccommodate a plurality of isolated spheroids in separated spheroidwells in a single chamber, preferably having, for example, one spheroidper spheroid well.

In embodiments, the disclosure provides a method of making an article,the article comprising:

a frame having a chamber, for example, a well, comprising:

-   -   an opaque side wall surface;    -   a top aperture;    -   a gas-permeable, transparent bottom surface; and    -   optionally a chamber annex surface having a second bottom remote        from the transparent bottom surface; and    -   at least a portion of the transparent bottom comprises at least        one concave arcuate surface, that is, a transparent rounded or        curved surface;

the method of making comprising:

combining the gas-permeable, transparent arcuate bottom surface portionand an opaque side wall surface portion to form the article.

The method of making the article can further comprise, for example,inserting a porous membrane in at least one of the chambers.

There are several methods that can be used to make an opaqueround-bottom multiwell plate having opaque sidewalls and a base with aclear window. The multiwell plate made by Examples 1 or 2, and havingflat well bottom geometry can be subsequently thermally-reformed tocreate an alternative well-bottom geometry. A multiwell plate having aflat well-bottom geometry can be, for example, positioned in a nest thathas at each well-bottom position areas removed from the nest that have acircular perimeter in roughly the same dimensions as the well format, orthe nest can have a tapered angle, for example, from about 30 to about60 degrees, including intermediate values and ranges, such as 40 to 50,and 45 degrees, turning into a radius at the apex, that is, rounded atthe very bottom of the well. A heated platen, heated for example at 180to 400 degrees F., more preferably 250 to 300 degrees F., having anarray of pins that have, for example, a matching 45 degree tapered anglefrom the sidewall sloping into a radius at the apex, is held stationary,with a pin positioned over each well. The pin-platen is actuated so thatthe pins descend into the wells of the multiwell plate to contact theclear flat well-bottom surface. Time, temperature, and pressure canchange the well-bottom material geometry as the pins advance until thereformed well-bottom material meets the matching nest geometry. Thearray of pins may be coated with a release agent, such as PTFE, toprevent the reformed well-bottom geometry from sticking to the heatedpins, or the pins can be uncoated. The nest may also have a vacuumassist on the nest to help draw the polymer into the well geometry.Cooling air can be injected into the top of the wells above the heatedpins to cool the hot pins slightly and facilitate their release from thepolymer of the reformed well-bottom geometry. The platen with the pinarray and the nest with the matching geometry can be rapidly changed tocomply with many multiple well formats, including 384- and 1536-wells.Any number of wells may be reformed at the same time. In embodiments,the pin-platen can be held stationary while the nest holding the insertcan be actuated to be brought into contact with the stationarypin-platen. In embodiments, both the pin-platen and the nest holding theinsert can be actuated to provide relative motion that achieves contactand closure between the pins and the wells of the insert.

A v-shaped well base geometry having, for example, a tapered angle of 45degrees, sloping from the well sidewall to a radius at the apex, issuperior for spheroid formation and superior for optical measurementcompared to a hemispherical well-bottom geometry. The v-shaped well basegeometry also permits overmolding on the well base geometry using thesame flat pins that can be used to over-mold opaque polymer onto a flatinsert. The 45 degree well base tapered angle permits the pins tocontact each well base on the bottom tapered sidewall. While this canalso be done with a hemispherical geometry, it is more difficult. If thepin is not able to shut-off on the sidewall, the opaque polymer can leakinto or onto the clear well bottom. Alternatively, if the pin cannotachieve shut-off on the sidewall, the pin can be made larger than thewell, and achieve shut-off on the flat area surrounding each well at thetop of the insert. This alternative solution is not desired because itcan create a flat ledge within each well upon which cells can settle.Cells that settle on the ledge cannot contribute to the spheroid in thecenter of the well bottom, but can cause large variation in experimentalresults.

In embodiments, the post-forming equipment can have, for example, aheated pin, such as a single pin, a row of pins, or an array of pins,that can match the number and alignment of, for example, a single wellor a plurality of wells in a row or an array of wells of an injectionmolded microwell plate. The geometry at the end of each pin can have,for example, a 45 degree angle or taper from the sidewall and have aradius at or near the apex.

In embodiments, an injection molded plate having a flat well-bottomgeometry can be situated and secured in a nest for the post-forming stepwhere the heated pin or pin array assembly is pressed, for example, intothe flat well-bottoms of an injection molded microplate. The nest canhave a matching 45 degree angle at each well position to match andreceive the pin shape. In embodiments, a platen can hold the heated pinor pins and the nest holds the matching well-bottom geometry. The planeof the pin platen and the plane of the nest can be aligned in paralleland both can be perpendicular to a common axis for proper reversible andreproducible pressing.

The platen, the nest, or both, can be rapidly interchanged toaccommodate other multiwell plate formats such as 384- and 1536-wellplates. The platen bearing the heated pin(s) can be pressed into amatching well(s) of the multiwell plate to form the selected geometrydefined by the geometry or cross-section of the pin's tip, such ashemispherical, conical, v-shaped, w-shaped, wedged shaped, and likegeometries, or combined geometries, in the clear base of the well.

In embodiments, the disclosure provides a method of culturing spheroidscomprising, for example:

charging any of the aforementioned articles with culture media; and

adding spheroid forming cells to the culture media.

Materials and Methods

There are several methods that can be used to make the disclosed articleor articles. In embodiments, the disclosed article can be, for example,a multiwell plate having an opaque side wall for the wells and having anarcuate or concave rounded base or bottom surface, the base or bottomsurface can be gas permeable and transparent or clear to visible light.

Commonly owned and assigned U.S. Pat. No. 7,674,346, mentions methods ofmaking well plates such as bonding a polymeric holey plate to a glass orpolymeric base and using, for example, heat or radiation to weld a gaspermeable thin film to a holey plate to form the bottom of microplates(see e.g., col 2, lines 4 to 44).

Commonly owned and assigned U.S. Pat. No. 5,759,494, mentions methods ofmaking well plates having opaque sidewalls by, for example, over-moldinginserts to prevent cross-talk. Polymethylpentene (TPX) of the presentlydisclosed gas permeable spheroid plate can be selected as a suitablebase or wall material that can be used in methods of making in the '494patent and the present disclosure.

In embodiments, the structure of the presently disclosed gas permeablespheroid plate can also include one or more ridges arranged in one ormore grid patterns as disclosed in the '494 patent, see for example,col. 4, line 30 to col. 5, line 54, to further reduce or preventcross-talk. In over-molded plates without a grid, the perimeter of thewell, a film, tended to peel away from the molded plate body. The gridappears to keep the film from peeling away from the plate body as it isreforming. The grid also appears to prevent stress marks from appearingin the film, since stress marks do appear in the plates without thegrid.

U.S. Pat. No. 6,811,752, mentions methods of making a device havingmicro-chambers and microfluidic flow, and gas permeable, liquidimpermeable membranes.

In one method (insert molding), the multiwell plate bottom or baseportion having at least one rounded, cupped, or dimpled impression inthe base is injection molded, or thermoformed using a clear polymer.Next, the molded or thermoformed clear multiwell bottom plate isinserted into a vertical press, then an opaque polymer is injectedforming the remainder of the multiwell plate onto the clear, round wellbottoms. An alternative to insert molding uses a 2-shot molding process.

Referring again to the Figures, FIG. 1 schematically shows methods forjoining preformed or molded components. An injection molded orthermoformed opaque, holey plate portion (110) of a multiwell plate(i.e., without well bottoms) is joined to the injection molded orthermoformed clear, round, well-bottom plate portion (120). This joiningto form the article (130) can be accomplished by any suitable method,for example, by placing the well-bottom plate (120) (or “insert”) into avertical press and molding the opaque holey plate portion (110) onto thewell-bottom plate portion. An alternative method of making can be, forexample, injection molding the entire multiwell plate using a 2-shotmolding process. The methods form an integral multiwell plate that hasclear, rounded-well bottoms and opaque sidewalls. Any method of joinerycan be used to attach preformed portions (110) and (120), including, forexample, adhesive, ultrasonic, thermal, IR, laser welding, and likemethods, or combinations thereof. Subsequent optional well coating with,for example, an ultra-low attachment material, and the force of gravity,can facilitate the cells self-assembling into a spheroid. Using a gaspermeable material of appropriate thickness for, at least, the clear,round or cupped well-bottom portion of the multiwell plate enhancesoxygen availability for the cells in the spheroid.

EXAMPLE(S)

The following examples serve to more fully describe the manner of usingthe above-described disclosure, and to further set forth best modescontemplated for carrying out various aspects of the disclosure. Theseexamples do not limit the scope of this disclosure, but rather arepresented for illustrative purposes. The working example(s) furtherdescribe(s) how to prepare the disclosed cell culture well articles.

Example 1 Injection Mold or Thermoform Methods

FIGS. 2A to 2C show an alternative method for creating an opaque“sleeve.” FIG. 2A shows an injection molded, or thermoformed wellportion of a multiwell plate (210) (no skirt) having rounded or cuppedwell bottoms made using a clear polymer. FIG. 2B shows an injectionmolded, or thermoformed second multiwell plate including the skirt (220)but without well bottoms made using an opaque polymer. The welldiameters of the opaque plate (220) are molded to be slightly largerthan the well diameters of the clear plate (210). The clear portion isslipped into the opaque portion and may be permanently attached via anumber of suitable methods including, for example, an adhesive,ultrasonic welding, IR welding, thermal welding, laser welding, and likemethods. FIG. 2C shows a compression fit of the clear plate into theopaque plate to provide the assembled plate article (230). The opaqueplate (220) acts as a sleeve structure to receive the clear platestructure and the combination forms the article having wells havingopaque sidewalls and a clear window at the arcuate or rounded base ofthe well.

FIG. 3 shows another alternative method of making the disclosed articleusing distortion printing. A clear polymer sheet (310) is selectivelyprinted with an opaque material to form printed sheet (315) having clearwindow regions or zones (330). The sheet can be selectively thermoformedto introduce the cups (i.e., the distortion) before or after printing sothat the clear zones (330) of the printed sheet (315) become the centerof the well bottoms. In embodiments, two halves, for example, a holeyplate having clear bottoms (350) and sheet (315) of a mold are used toform the complete multiwell plate (340) from the distortion printedpolymer sheet. Once thermoformed into a multiwell plate (360), all ofthe plate will appear opaque except for the clear zone (370) in thecupped-shaped well bottoms that form a window for optical viewing at orfrom the well bottom.

Example 2

In another method of making, one can mold or thermoform an opaquemultiwell plate that has no well-bottoms (i.e., make an opaque “holeyplate”). Next, one can injection mold, or thermoform the multiwell platebottom or base portion using a clear polymer. Then one can attach theholey plate and base components together using any of various assemblyprocesses, including, for example, adhesive, ultrasonic, IR, thermal,laser welding, and like processes.

Example 3

In another method of making, one can injection mold, or thermoform theentire well of a multiwell plate (without a skirt) using a clearpolymer. Next, one can injection mold, or thermoform another multiwellplate (including the skirt) but without well bottoms using an opaquepolymer. The well diameters of the opaque plate are molded to beslightly larger than the well diameters of the clear plate. The opaqueplate acts as a sleeve to receive the clear plate and thus form opaquesidewalls and with a clear window at the base of the well.

Example 4

In another method of making, one can distortion print an opaque coatingon the exterior of a clear polymer sheet, leaving a clear zone in thearea that will be thermoformed with, for example, a cup or dimple, ormultiples thereof, into the center of the well bottom. Once thermoformedinto a multiwell plate, all of the plate will appear opaque except forthe clear zone that forms a window for optical viewing.

Example 5 Non-Adhesive Coating

The interior well-bottom surface, walls, or both can optionally be madenon-adhesive to cells by coating those surfaces with a polymer that doesnot have any characteristics that promote cell attachment, such as aperfluorinated polymer, olefin, or like polymers, and mixtures thereof.Alternatively, following assembly of the opaque multiwell plate withclear, round or cupped well-bottoms, the interior well-bottom may becoated with a non-binding material such as an ultra-low attachmentmaterial, agarose, nonionic hydrogel, or like materials, that caninhibit cell attachment. Coatings can also be applied to the clear wellbottom portion of the multiwell plate prior to joining with the opaqueportion. The combination of, for example, a low-attachment substrate,the well curvature in the body and the base portions, and gravity, caninduce cells to self-assemble into spheroids, which cell clusters areknown to maintain differentiated cell function indicative of a morein-vivo like response (see Messner, et al., Archives of Toxicology, Nov.11, 2012). Perfluorinated polymers or polymers such as poly4-methylpentene also provide gas permeability at thicknesses normallyused in molding processes, which gas permeability can be beneficial formetabolically active cell types. Representative thickness and ranges ofgas permeable polymer can be, for example, from about 0.001 inch toabout 0.025 inch, from 0.0015 inch to about 0.03 inch, includingintermediate values and ranges (where 1 inch=25,400 microns; 0.000039inches=1 micron). Additionally or alternatively, other materials havinghigh gas permeabilities, such as polydimethylsiloxane polymers, canprovide sufficient gas diffusion at a thickness, for example, of up toabout 1 inch.

FIGS. 9A and 9B show a cross-section view of the combination (900) ofthe injection molding pin shut-off in contact with a well-bottom havingdifferent geometries (9A; v-shaped, and 9B; hemisphere shaped). FIG. 9Aschematically demonstrates how the pin (910), which fits inside thewell, can shut-off (912) on the 45 degree angle of the v-shapedwell-bottom geometry (915). FIG. 9B schematically demonstrates how thepin (910) can encounter potential difficulty of achieving shut-off (912)on the hemispherical well-bottom geometry (920).

FIGS. 10A and 10B show cross-section views of an alternative design fora pin shut-off. FIGS. 10A and 10B demonstrate the utility of an optionaldistortion collar (1010). The wall thickness of the distortion collar(1010) can be, for example, from about 0.005 to 0.020 inches, and can bedetermined by, for example, the modulus of the polymer, and thegeometric constraints of the multiwell plate. The distortion collarsurrounds or brackets the well and is able to distort, i.e., resilientlyflex and stretch, under the pressure of the injection molding pin (910)to shut-off, that is, to seal or seal-off the contoured transparent baseportion from the injected opaque resin for the side walls. FIG. 10Ashows a pin (910) centered in the well and on a common axis (1011) withthe pin. FIG. 10B shows the pin slightly offset (1015) from the originalcommon axis (1011) and pin axis (1012), for example as shown, to theleft of center. The distortion collar permits the pin to stilladequately shut-off or be sealed to prevent the flow of opaque side wallpolymer into the clear well-bottom. The distortion collar yields to agreater extent to the left side and yields to a lesser extent on theright side as illustrated by the respective force vector arrows (1016,1017).

Example 6

Post Forming Method Another method of making a well plate article of thedisclosure is accomplished by thermal reforming. In the thermalreforming method a commercially available plate, for example, havingstandard black (opaque) side-walls and a clear flat plastic bottom isselected. Using a combination of vacuum on the outside and pressuresupplied by a hot forming tool with a full radius pushed into theinterior of the well(s) on the opposite side of the plate and furtherinto the bottom of the well, the concave arcuate base or tapered basehaving a round bottom radius is formed in the well bottom. The outsidebottom area can be, for example, preheated with IR radiation tofacilitate forming the cupped or internal concave arcuate bottom radius,or the tapered base.

Example 7 Method of Use; Culturing of Cells

FIGS. 11A to 11D show images of cell spheroids in different well-bottomgeometries. The images were captured on a light microscope at 20×magnification (the reference scale equals 1,000 micrometers).

Image 11A shows a spheroid in an all clear control 96-well plate (i.e.,the walls of this comparative plate are transparent) having a fullhemisphere well-bottom geometry.

Image 11B shows a spheroid in a prototype thermally reformed 96-wellplate having a v-shaped or 45 degree tapered well-bottom geometry.

Image 11C shows a spheroid in an injection-molded single well with thev-shaped or 45 degree well-bottom geometry.

Image 11D shows a spheroid in an injection-molded single well having afull hemisphere well-bottom geometry.

HT-29 cells were seeded into wells including the comparative well (11A)and inventive v-shaped wells (11B and 11C) and hemisphere shaped well(11D) at a concentration of 10,000 cells per well, and incubated for 96hours at 37 degrees C. in an incubator with 5% CO₂ and 85% humidity. TheFIGS. 11A to 11D images were captured at 20× magnification on a lightmicroscope after 96 hours of incubation.

The cell seeding procedure generally included the following steps:trypsinize; count the cells; centrifuge to remove medium from the cells;and re-suspend the cells. Seeding densities can be, for example: 10 k,20 k or 30 k cells per well. The seeding volume can be, for example, 200microliters per well. Monitoring images can be recorded, for example, at30 minutes, at 24 hrs, and at 96 hrs after seeding, includingintermediate values and ranges.

The spheroids in images 11B and 11C are in the center of the v-shapedwell base. The spheroids in images 11A and 11D are not in the center ofthe hemisphere. The v-shaped base geometry appears to promote centeringof the spheroids in the center of the well base, which centeringfacilitates optical visualization.

In embodiments, one can form a gas permeable, rounded well-bottom usingthe thermal reforming process. In existing methods, the plates can bemolded on flat inserts of, for example, polystyrene that is 0.005″,which thickness is considered not gas permeable. However, during thethermal reforming process, one can decrease the thickness of the insertat the radiused apex to, for example, about 0.003″ (photograph notincluded), which makes the well-bottom as gas permeable as theHYPER-product film (which is also 0.003″ thick polystyrene).

The disclosure has been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications are possible while remaining within thescope of the disclosure.

1-19. (canceled)
 20. A cell culture article comprising: a chambercomprising: a side wall a top aperture; and a gas-permeable , liquidimpermeable bottom comprising at least one concave surface, wherein atleast a portion of the bottom is transparent and wherein at least aportion of the bottom comprises a low-adhesion or no-adhesion materialin or on the at least one concave surface.
 21. The article of claim 20further comprising a liner having a porous membrane situated within aportion of the chamber to divide the chamber into an upper chamber and alower chamber.
 22. The article of claim 20 further comprising a chamberannex for receiving a pipette tip for aspiration, the chamber annexcomprises a surface adjacent to and in fluid communication with thechamber, the chamber annex having a second bottom spaced away and at anelevation above the gas-permeable, transparent bottom, wherein thesecond bottom deflects fluid dispensed from a pipette away from thetransparent bottom.
 23. The article of claim 22 further comprising aliner having a porous membrane situated within a portion of the chamberto form an upper chamber and a lower chamber the porous membraneproviding separation of a second cellular material in the upper chamber,from a first cellular material in the lower chamber .
 24. The article ofclaim 20 wherein the article comprises from 1 to about 2,000 of saidchambers, wherein each chamber is physically separated from any otherchamber.
 25. The article of claim 20 wherein the at least one concavesurface comprises a plurality of concave surfaces within the samechamber.
 26. The article of claim 20 wherein the at least one concavesurface comprises a hemispherical surface, a conical surface having ataper of 30 to about 60 degrees from the side walls to the bottom, or acombination thereof.
 27. The article of claim 20 wherein the side wallsurface comprises a vertical cylinder, a portion of a vertical conic ofdecreasing diameter from the chamber's top to bottom surface, a verticalsquare shaft having a conical transition to the at least one concavebottom surface, or a combination thereof
 28. The article of claim 20wherein the at least one concave surface comprises a hemisphere, or aportion thereof, having a diameter of from about 250 to about 5,000microns.
 29. A perfusion plate apparatus comprising: at least one cellculture article of claim 20; a media source well in fluid communicationwith at least one chamber of the cell culture article that controllablyprovides a source fresh media to the at least one chamber; and a wastewell in fluid communication with the at least one chamber of the cellculture article that controllably receives waste media from the at leastone chamber.
 30. The apparatus of claim 29 further comprising a porousliner situated within a portion of the at least one chamber to form anupper chamber and a lower chamber, the porous liner providing separationof a second cellular material in the upper portion of the chamber, froma first cellular material in the lower chamber.
 31. The apparatus ofclaim 29 further comprising: a perfusion plug between the source welland the at least one chamber, a perfusion plug between the at least onechamber and the waste well, or both.
 32. The apparatus of claim 29wherein the at least one concave surface comprises a plurality ofadjacent concave arcuate surfaces.
 33. A method of making the article ofclaim 20 comprising: combining the gas-permeable, transparent, bottomportion and a side wall surface portion to form the article.
 34. Themethod of claim 33 wherein combining is accomplished with: injectionmolding, thermal reforming, post forming, distortion printing, overmolding, or combinations thereof.
 35. The method of claim 33 wherein thegas-permeable, transparent, bottom portion includes a distortion collarintegral with or attached to the gas permeable transparent bottomportion to prevent side wall material from entering the arcuate bottomportion.
 36. The method of claim 33 further comprising inserting aporous membrane in at least one of the chambers of the resultingarticle.
 37. A method of culturing spheroids comprising: charging thearticle of claim 20 with culture media; and adding spheroid formingcells to the culture media.
 38. The method of claim 37 furthercomprising optically examining the cultured spheroid forming cellsthrough the transparent bottom.
 39. The article of claim 20, furthercomprising culture media and a spheroid in said chamber.
 40. The articleof claim 20, wherein said side wall is adjacent to said transparentportion of said bottom.
 41. The article of claim 20, further comprisingone or more auxiliary chambers in fluid communication with said chamber.